High resolution 3-d spectral domain optical imaging apparatus and method

ABSTRACT

Methods and apparatus are presented for obtaining high-resolution 3-D images of a sample over a range of wavelengths, optionally with polarisation-sensitive detection. In preferred embodiments a spectral domain OCT apparatus is used to sample the complex field of light reflected or scattered from a sample, providing full range imaging. In certain embodiments structured illumination is utilised to provide enhanced lateral resolution. In certain embodiments the resolution or depth of field of images is enhanced by digital refocusing or digital correction of aberrations in the sample. Individual sample volumes are imaged using single shot techniques, and larger volumes can be imaged by stitching together images of adjacent volumes. In preferred embodiments a 2-D lenslet array is used to sample the reflected or scattered light in the Fourier plane or the image plane, with the lenslet array suitably angled with respect to the dispersive axis of a wavelength dispersive element such that the resulting beamlets are dispersed onto unique sets of pixels of a 2-D sensor array.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/951198 filed 12 Apr. 2018 which is a continuation of U.S.Pat. No. 9,955,863 dated 1 May 2018, the entire contents of which areincorporated herein by reference. The present application claimspriority from Australian Provisional Patent Application No 2015901970entitled ‘High resolution 3-D spectral domain optical imaging apparatusand method’ filed on 28 May 2015, the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to optical imaging apparatus and methods, and inparticular to a 3-D spectral domain optical coherence tomography (OCT)apparatus with full range and extended depth of focus that samples thecomplex field. However it will be appreciated that the invention is notlimited to this particular field of use.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should inno way be considered as an admission that such prior art is widely knownor forms part of the common general knowledge in the field.

Optical coherence tomography (OCT) is a widely used interferometrictechnique for studying biological samples including in vivo tissue suchas the human eye, with lateral and depth resolution, using informationcontained within the amplitude and phase of reflected or scatteredlight. OCT systems generally utilise a Michelson interferometerconfiguration, with two main approaches being employed: time domain OCTand spectral domain OCT.

In time domain OCT coherence properties of a partially coherent sourcesuch as a superluminescent light emitting diode (SLED) with a coherencelength of several microns are utilised by interfering light reflectedfrom a sample with a reference beam provided by the same source, butwith a time-varying path length. At a specific depth in the samplecorresponding to the path length delay in the reference arm, aninterference envelope of fringes will be detected in the combinedback-reflected signal, allowing the reflection profile in the depthdimension to be reconstructed. Commonly this is done for only a singlesample point at a time, and the corresponding scan of depth is known asan ‘A-scan’.

Instead of scanning a delay line, spectral domain OCT techniques analysethe reflected light by interfering it with a reference beam, either as atime-varying function of wavelength (swept source OCT) or by dispersingthe different wavelengths with a grating or other spectral demultiplexerand detecting them simultaneously along a detector array. The spectraldomain information is the Fourier transform of the spatial (depth)reflection profile, so the spatial profile can be recovered by a FastFourier Transform (FFT). Generally speaking, spectral domain OCT systemsare preferred over time domain OCT systems because they have a ˜20 to 30dB sensitivity advantage.

OCT techniques can be adapted to provide a laterally resolved ‘B-scan’by scanning the sample beam relative to the sample in one axis, or a‘C-scan’ by scanning in two axes. Faster acquisition is generallydesirable irrespective of the type of scan, especially for reducingmotion-induced artefacts with in vivo samples, and has been greatlyimproved over the previous 20 to 25 years by advances in several fieldsincluding faster swept source scanning rates and photodetector arrayreadout speeds. However a fundamental limitation with scanning spotschemes, especially for in vivo applications, is presented by lasersafety regulations: reducing dwell time to increase scanning speedwithout being able to increase the applied power will inevitably degradethe signal to noise ratio.

Consequently there has also been research into ‘parallelised’ OCTsystems in which an extended sample area is probed with lateralresolution, or an array of sample spots probed simultaneously. It isrelatively straightforward to parallelise time domain OCT, e.g. byutilising a CCD camera and imaging optics as described in U.S. Pat. No.5,465,147 entitled ‘Method and apparatus for acquiring images using aCCD detector array and no transverse scanner’. This provides a twodimensional (2-D) en face image, with depth resolution provided byscanning the reference mirror as usual in time domain OCT.

Swept source spectral domain OCT can be parallelised in similar fashion,as described in Bonin et al ‘In vivo Fourier-domain full-field OCT ofthe human retina with 1.5 million A-lines/s’, Optics Letters 35(20),3432-3434 (2010). However because each frame corresponds to a singlewavelength, the acquisition time for each A scan is equal to the frameperiod times the number of k-points (wavelength samples) acquired. Evenfor very high speed cameras with frame rates of 100s of kHz, this canlead to A scan acquisition times of many ms which can lead to motionartefacts especially with in vivo samples. PCT patent application NoPCT/AU2015/050788, entitled ‘Multichannel optical receivers’, disclosesan alternative parallelised swept source OCT scheme that enables fasteracquisition. In one particular implementation a plurality of spots on asample are illuminated simultaneously and the reflected or scatteredsignal light mixed with a reference beam to form a plurality ofinterferograms with unique carrier frequencies.

Parallelised spectrometer-based spectral domain OCT enables single shotB-scan acquisition, although existing schemes are limited by the factthat one axis of a 2-D photodetector array is occupied by the wavelengthdispersion. In a configuration described in published US patentapplication No 2014/0028974 A1 entitled ‘Line-field holoscopy’,cylindrical lenses are used to produce a line illumination on a sampleand on a reference mirror. As shown schematically in FIG. 1, thecombined return sample and reference beams from a line illumination 2are dispersed with a dispersive element such as a grating 4 and detectedwith a 2-D sensor array 6. A Fourier transform along the spectral axis 8provides an A-scan for each position 9 along the illuminated line 2. Forfull three-dimensional (3-D) imaging the illuminated line ismechanically scanned in the orthogonal direction and the 2-D sensorarray read out repeatedly.

Even if a linear B-scan of a sample is sufficient, i.e. 3-D imagingisn't required, a scan in the orthogonal direction may still benecessary, e.g. for digital wavefront correction to correct for lensaberrations and the like, or to provide increased depth of field.Furthermore for these purposes the repeated linear scans have to bephase coherent, which is generally difficult.

It is generally preferred for spectral domain OCT apparatus to beconfigured to sample the unambiguous complex field of the interferencesignal, rather than just the detected real-valued interference signal,to distinguish positive and negative path length delays and thereforeenable imaging over the full depth of field range. A variety ofapproaches for capturing the complex field have been described. Forexample Jungwirth et al ‘Extended in vivo anterior eye-segment imagingwith full-range complex spectral domain optical coherence tomography’,Journal of Biomedical Optics 14(5), 050501 (2009) describes, for ascanning spot scheme, a solution in which the sample phase is ditheredas the sample is scanned. A key drawback of this approach is that samplemovement can cause loss of phase coherence during scanning. Line fieldsystems, which have improved phase stability, have been described whichdo not require dithering of the sample phase. In US 2014/0028974 A1 forexample the complex field is obtained by sampling the signal in the farfield of a linear illumination, whilst in Huang et al ‘Full-rangeparallel Fourier-domain optical coherence tomography using a spatialcarrier frequency’, Applied Optics 52(5), 958-965 (2013), the line fieldis captured in the image plane, with an off-axis reference providingaccess to the complex field.

The transverse resolution of an OCT apparatus is determined, for a givenwavelength, by the numerical aperture of the objective lens. Howeverincreasing the numerical aperture of the objective invariably reducesthe depth of field, resulting in a trade-off between transverseresolution and depth of field. A variety of software-based or digitalfocusing techniques have been proposed to overcome this trade-off toincrease the depth of field. These approaches generally assume that thephase coherence between scattering points is maintained during scanningand sample collection, and the field may be captured in the image planeor the Fourier plane.

In one example, synthetic aperture techniques are discussed in Mo et al‘Depth-encoded synthetic aperture optical coherence tomography ofbiological tissues with extended focal depth’, Optics Express 23(4),4935-4945 (2015). In another example, the forward model (FM) approach ofKumar et al ‘Numerical focusing methods for full field OCT: a comparisonbased on a common signal model’, Optics Express 22(13), 16061-16078(2014), involves sampling the 3-D captured interferometric signalI(x,y,k) in the image plane using a full field swept source OCTapparatus with a 2-D CMOS camera. An unambiguous phase is obtained byrequiring the sample to be on one side only of the zero delay, and thedefocus correction is achieved by applying a numerical phase correctionbased on a Fresnel wavefront propagation model. This numerical phasecorrection is achieved by first performing a 1-D FFT of the real valuedsignal along the spectral axis to give the complex field, I(x, y,k)→E(x, y, Δz). This is followed by a 2-D FFT of the lateral coordinatesfor all positive delays, E(x, y, Δz)→E(k_(x), k_(y), Δz). The Fresnelcorrection for defocus correction is then applied: E(k_(x), k_(y),Δz)→E(k_(x), k_(y), Δz)γ, where

$\gamma = {{\exp \left( {i\frac{\lambda_{0}\Delta \; {zM}^{2}}{4\pi \; n}\left( {k_{x}^{2} + k_{y}^{2}} \right)} \right)}.}$

Here, the wavelength is replaced by the centre wavelength λ₀, n is therefractive index of the sample and M is the magnification of the OCTapparatus. A 2-D inverse FFT (IFFT) with respect to the spatialfrequencies of the phase-corrected field gives an image focused over thefull volume.

Digital focusing with a full-range line-field OCT system has beendemonstrated in Fechtig et al ‘Full range line-field parallel sweptsource imaging utilizing digital refocusing’, Journal of Modern Optics(2014), DOI: 10.1080/09500340.2014.990938. In this case the sample fieldis measured in the image plane and full range measurements are achievedby using an off-axis configuration of the reference arm. This off-axisconfiguration introduces a lateral carrier frequency which shifts theinterference term in frequency space enabling the positive and negativefrequency components to be separated, thereby enabling measurement ofthe complex signal. Phase noise in the scanning direction restricts thedigital focusing to one dimension, which is applied to each successive Bscan. The complex signal is obtained by first taking a 1-D FFT along thespatial axis corresponding to the off-axis reference, after which afilter can be applied to select the positive frequency signal componentfrom its complex conjugate artefact and the non-interferometricbackground. A 1-D IFFT then gives a signal measurement with unambiguousphase. Digital focusing is achieved by performing a 1-D FFT along thespectral axis followed by a 1-D FFT of the lateral coordinates to giveE(k_(x), Δz), where Δz now extends over the full range. Multiplicationby the 1-D phase correction factor followed by a 1-D IFFT gives thefocused B-scan over the full range.

A full-field swept source OCT system with sampling in the far field isdescribed in Hillmann et al ‘Holoscopy—holographic optical coherencetomography’, Optics Letters 36(13), 2390-2392 (2011). In this system,2-D interferograms for each wavelength are propagated to a specificdelay Δz. A 1-D FFT along the spectral axis is then used to reconstructthe focused object for this depth Δz. This process is repeated for arange of delays and the refocused regions are then stitched together.Full range imaging with sampling in the Fourier plane has beendemonstrated using an off-axis reference beam to obtain an unambiguousphase, as described in Hillmann et al ‘Efficient holoscopy imagereconstruction’, Optics Express 20(19), 21247-21263 (2012). Thisnumerical post-processing approach, in which the 3-D signal isinterpolated onto a non-equally spaced grid, provides a volume imagewith a resolution equivalent to the focal plane resolution throughout anextended portion of the volume. A final 3-D FFT then gives the focusedvolume image. Similar methods are used in inverse synthetic aperturemicroscopy (ISAM), described for example in Ralston et al‘Interferometric synthetic aperture microscopy’, Nature Physics 3(2),129-134 (2007).

We note that the approaches described above assume a simple model fordepth-dependent defocus. An alternative approach that compensates forunknown optical aberrations using sub-aperture correlations is describedin Kumar et al ‘Subaperture correlation based digital adaptive opticsfor full field optical coherence tomography’, Optics Express 21(9),10850-10866 (2013).

An important limitation of full-field OCT systems, compared topoint-scanning systems, is that that they are susceptible to crosstalkfrom multi-path scattering and hence have reduced sensitivity. Inaddition, the lack of confocal filtering increases the susceptibility tospurious reflections from outside the coherence length of the system.The line field approach of US 2014/0028974 A1 partially alleviates theselimitations compared to that of a full field system by confocal gatingin one axis. An alternative approach to mitigating crosstalk is to use aspatially incoherent source.

Unless the context clearly requires otherwise, throughout thedescription and the claims the words ‘comprising’, ‘comprises’ and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense. That is, they are to be construed in thesense of ‘including, but not limited to’.

OBJECT OF THE INVENTION

It is an object of the present invention to overcome or ameliorate atleast one of the limitations of the prior art, or to provide a usefulalternative. It is an object of the present invention in a preferredform to provide spectral domain OCT apparatus and methods for acquiring3-D images of a sample employing single shot acquisition techniques. Itis another object of the present invention in a preferred form toprovide apparatus and methods for obtaining improved high resolutionoptical images of a retina based on numerical reconstruction of thespectral characteristics of light reflected or scattered from a smallvolume of the retina, with correction of aberrations present in thesample eye.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is providedan apparatus for retinal imaging, said apparatus comprising:

-   (i) a multi-wavelength optical source;-   (ii) an angularly variable illumination system for directing at    least two portions of light emitted from said optical source onto    each of two or more volumes of the retina of a sample eye;-   (iii) a measurement system for receiving signals of light reflected    or scattered from each of said two or more volumes, each said signal    being a function of the phase and amplitude of the electric field    vector of the reflected or scattered light, and for making    simultaneous measurements over a range of wavelengths for each of    said signals; and-   (iv) a processor for processing the measurements to generate one or    more numerical representations of an optical characteristic of said    retina over said two or more volumes, and to create from said one or    more numerical representations a three-dimensional composite image    over a region of said retina comprising at least a portion of said    two or more volumes.

According to a second aspect of the present invention there is providedan apparatus for imaging a sample, said apparatus comprising:

-   (i) a multi-wavelength optical source;-   (ii) an illumination system for sequentially directing at least two    portions of light emitted from said optical source onto each of two    or more volumes of a sample, said sample being located at or close    to a focal plane of an optical power element of said apparatus;-   (iii) a measurement system for receiving signals of light reflected    or scattered from each of said two or more volumes, each said signal    being a function of the phase and amplitude of the electric field    vector of the reflected or scattered light, and for making    simultaneous measurements over a range of wavelengths for each of    said signals; and-   (iv) a processor for processing the measurements to generate one or    more numerical representations of an optical characteristic of said    sample over said two or more volumes, and to create from said one or    more numerical representations a three-dimensional composite image    of said sample over a region comprising at least a portion of said    two or more volumes.

The first and second aspects share a number of preferments. Preferably,the processor is adapted to create the three-dimensional composite imageusing digital refocusing or digital correction of aberrations of thesample eye or of the sample. In certain embodiments the processor isadapted to generate numerical representations of the opticalcharacteristic over each of the two or more volumes, and to create thethree-dimensional composite image from the numerical representations. Inother embodiments the processor is adapted to generate a numericalrepresentation of the optical characteristic over the two or morevolumes, and to create the three-dimensional composite image from thenumerical representation.

In certain embodiments the illumination system is adapted tosequentially direct the at least two portions of light onto the two ormore volumes of the retina. In other embodiments the illumination systemis adapted to simultaneously direct the at least two portions of lightonto the two or more volumes of the retina.

The measurement system preferably comprises a two-dimensional lensletarray for sampling the signals and a wavelength dispersive element fordispersing the sampled signals onto a two-dimensional sensor array,wherein the lenslets of the lenslet array are positioned with respect tothe wavelength dispersive element such that, in use, each of the sampledsignals is dispersed onto a set of pixels of the sensor array. Incertain embodiments the two-dimensional lenslet array is positioned soas to sample the signals in the Fourier plane. Preferably, thetwo-dimensional lenslet array comprises a rectilinear array of lensletsangled with respect to the dispersive axis of the wavelength dispersiveelement.

Preferably, adjacent pairs of the two or more volumes are partiallyoverlapping. In certain embodiments the processor is adapted to reducethe three-dimensional composite image to a high resolution B scan of theretina or sample.

In certain embodiments the optical characteristic is selected from thegroup comprising phase, reflectivity, refractive index, refractive indexchanges and attenuation. In certain embodiments the measurement systemis adapted to capture phase and amplitude information for at least firstand second polarisation states of the signals. In these embodiments theoptical characteristic may comprise birefringence or degree ofpolarisation.

For each of the two or more volumes the illuminated surface of theretina or sample is preferably less than or equal to 500 μm×500 μm inarea, more preferably less than or equal to 200 μm×200 μm in area.

According to a third aspect of the present invention there is provided arelative phase-sensitive optical coherence tomography apparatuscomprising:

-   (i) an imaging system for acquiring first and second images of an    optical characteristic of a region of a sample in three spatial    dimensions, each said image comprising phase and amplitude    information over a range of wavelengths and each being acquired in a    single exposure, said second image being acquired a predetermined    time period after said first image; and-   (ii) a processor for:    -   (a) registering said first image to said second image to        determine a spatially resolved phase shift caused by motion or        distortion of said sample in any spatial dimension; and    -   (b) determining from said phase shift at least a component of        the displacement of said sample associated with said motion or        distortion.

The processor is preferably adapted to determine from the phase shiftand the predetermined time period a rate of displacement of the sampleassociated with the motion or distortion. In certain embodiments theprocessor is adapted to measure strain associated with the distortion ofthe sample, or to perform elastography measurements on the sample.

According to a fourth aspect of the present invention there is provideda polarisation-sensitive optical coherence tomography apparatuscomprising:

-   (i) an illumination system comprising a multi-wavelength optical    source for illuminating a volume of a sample with light of at least    a first polarisation state;-   (ii) an optical splitter for directing a portion of light reflected    or scattered from said sample away from said optical source;-   (iii) a measurement system for making a first set of simultaneous    measurements over a range of wavelengths, for at least first and    second polarisation states, of a signal of light reflected or    scattered from said sample, said signal being a function of the    phase and amplitude of the electric field vector of the reflected or    scattered light; and-   (iv) a processor for processing said first set of simultaneous    measurements to generate a three-dimensional representation of one    or more polarisation properties of the illuminated volume of said    sample.

Preferably, the one or more polarisation properties comprisesbirefringence or degree of polarisation.

In certain embodiments the illumination system is adapted tosubsequently illuminate the volume of the sample with light of a secondpolarisation state, different from the first polarisation state, and themeasurement system is adapted to make a second set of simultaneousmeasurements over a range of wavelengths. In these embodiments theprocessor is preferably adapted to process the first and second sets ofsimultaneous measurements to generate a three-dimensional representationof one or more polarisation properties of the illuminated volume of thesample.

In preferred embodiments the optical splitter comprises a polarisationindependent beam splitter. Preferably, the optical splitter comprises anapertured reflector having a total internal reflection surface and oneor more apertures that locally disrupt the total internal reflection atthe surface, for allowing transmission of light for illuminating thesample. More preferably, the apertured reflector comprises two totalinternal reflection surfaces spaced apart by one or more localised indexmatching regions that form the one or more apertures.

According to a fifth aspect of the present invention there is providedan optical coherence tomography apparatus for imaging a sample over anextended depth of field, said apparatus comprising:

-   (i) an illumination system adapted to illuminate, with beams    incident at two or more incident angles and each having at least    first and second wavelengths, a volume of a sample to be imaged in    three spatial dimensions;-   (ii) an interferometer adapted to measure, over said at least said    first and second wavelengths, and in a single shot at least for each    incident angle, a two-dimensional grid of sampling points of the    phase and amplitude of light reflected or scattered from the volume    of the sample illuminated at said two or more incident angles; and-   (iii) a processor for: registering and stitching together in the    Fourier Domain the measurements from the two or more incident angles    to create an extended Fourier Field of measurements; and generating    a three-dimensional image of an optical characteristic of the sample    by Fourier Transformation or digital processing of the extended    Fourier Field measurements.

In certain embodiments the illumination system is adapted to illuminatethe sample volume sequentially with the beams incident at two or moreincident angles. In alternative embodiments the illumination system isadapted to illuminate the sample volume simultaneously with the beamsincident at two or more incident angles.

In preferred embodiments the interferometer comprises: a two-dimensionallenslet array for providing the two-dimensional grid of sampling points;a two-dimensional sensor array; and a wavelength dispersive element fordispersing the light from each of the sampling points onto the sensorarray, wherein the lenslets of the lenslet array are positioned withrespect to the wavelength dispersive element such that, in use, thelight from each of the sampling points is dispersed onto a set of pixelsof the sensor array. In certain embodiments the two-dimensional lensletarray is positioned so as to sample the signals in the Fourier plane.The two-dimensional lenslet array preferably comprises a rectilineararray of lenslets angled with respect to the dispersive axis of thewavelength dispersive element. In preferred embodiments the lateralresolution of the three-dimensional image is enhanced by the extendedFourier Field measurements.

According to a sixth aspect of the present invention there is provided ahigh resolution optical imaging apparatus, comprising:

-   (i) an illumination system for illuminating, with a multi-wavelength    optical beam, a volume of a sample to be imaged in three spatial    dimensions;-   (ii) a sampling system for sampling in the Fourier plane light    reflected or scattered from the illuminated volume of said sample;-   (iii) a measurement system for simultaneous capture of phase and    amplitude information over a range of wavelengths of the sampled    reflected or scattered light; and-   (iv) a processor for processing the phase and amplitude information    to construct a three-dimensional image of an optical characteristic    of said sample over said illuminated volume.

In a preferred form the processor is adapted to construct thethree-dimensional image using digital refocusing or digital correctionof aberrations of the sample.

In preferred embodiments the measurement system comprises a wavelengthdispersive element for dispersing the sampled signals obtained from thesampling system onto a two-dimensional sensor array, wherein thesampling system is positioned with respect to the wavelength dispersiveelement such that, in use, each of the sampled signals is dispersed ontoa set of pixels of the sensor array. The sampling system preferablycomprises a two-dimensional lenslet array for sampling the reflected orscattered light to provide a two-dimensional grid of sampling points.

In certain embodiments the optical characteristic is selected from thegroup comprising phase, reflectivity, refractive index, refractive indexchanges and attenuation. In certain embodiments the measurement systemis adapted to capture phase and amplitude information for at least firstand second polarisation states of the reflected or scattered light. Inthese embodiments the optical characteristic may comprise birefringenceor degree of polarisation.

The illuminated surface corresponding to the illuminated volume ispreferably less than or equal to 500 μm×500 μm in area, more preferablyless than or equal to 200 μm×200 μm in area. In preferred embodimentsthe three-dimensional image has a spatial resolution of 3 μm or better.

According to a seventh aspect of the present invention there is provideda method for imaging the retina of a sample eye, said method comprisingthe steps of:

-   (i) providing a multi-wavelength optical beam;-   (ii) directing, with an angularly variable illumination system, at    least two portions of said multi-wavelength optical beam onto each    of two or more volumes of the retina of a sample eye;-   (iii) receiving signals of light reflected or scattered from each of    said two or more volumes, each said signal being a function of the    phase and amplitude of the electric field vector of the reflected or    scattered light;-   (iv) making simultaneous measurements over a range of wavelengths    for each of said signals; and-   (v) processing the measurements to generate one or more numerical    representations of an optical characteristic of said retina over    said two or more volumes, and to create from said one or more    numerical representations a three-dimensional composite image over a    region of said retina comprising at least a portion of said two or    more volumes.

According to an eighth aspect of the present invention there is provideda method for imaging a sample, said method comprising the steps of:

-   (i) providing a multi-wavelength optical beam;-   (ii) sequentially directing at least two portions of said    multi-wavelength optical beam onto each of two or more volumes of a    sample;-   (iii) receiving signals of light reflected or scattered from each of    said two or more volumes, each said signal being a function of the    phase and amplitude of the electric field vector of the reflected or    scattered light;-   (iv) making simultaneous measurements over a range of wavelengths    for each of said signals; and-   (v) processing the measurements to generate one or more numerical    representations of an optical characteristic of said sample over    said two or more volumes, and to create from said one or more    numerical representations a three-dimensional composite image of    said sample over a region comprising at least a portion of said two    or more volumes.

According to a ninth aspect of the present invention there is provided amethod for performing relative phase-sensitive optical coherencetomography measurements of a sample, said method comprising the stepsof:

-   (i) acquiring first and second images of an optical characteristic    of a region of a sample in three spatial dimensions, each said image    comprising phase and amplitude information over a range of    wavelengths and each being acquired in a single exposure, said    second image being acquired a predetermined time period after said    first image;-   (ii) registering said first image to said second image to determine    a spatially resolved phase shift caused by motion or distortion of    said sample in any spatial dimension; and-   (iii) determining from said phase shift at least a component of the    displacement of said sample associated with said motion or    distortion.

According to a tenth aspect of the present invention there is provided amethod for performing polarisation-sensitive optical coherencetomography measurements of a sample, said method comprising the stepsof:

-   (i) illuminating a volume of a sample with multi-wavelength light of    at least a first polarisation state;-   (ii) directing a portion of light reflected or scattered from said    sample away from the source of said multi-wavelength light;-   (iii) making a first set of simultaneous measurements over a range    of wavelengths, for at least first and second polarisation states,    of a signal of light reflected or scattered from said sample, said    signal being a function of the phase and amplitude of the electric    field vector of the reflected or scattered light; and-   (iv) processing said first set of simultaneous measurements to    generate a three-dimensional representation of one or more    polarisation properties of the illuminated volume of said sample.

According to an eleventh aspect of the present invention there isprovided a method for performing optical coherence tomography imaging ofa sample over an extended depth of field, said method comprising thesteps of:

-   (i) illuminating, with beams incident at two or more incident angles    and each having at least first and second wavelengths, a volume of a    sample to be imaged in three spatial dimensions;-   (ii) measuring interferometrically, over said at least said first    and second wavelengths, and in a single shot at least for each    incident angle, a two-dimensional grid of sampling points of the    phase and amplitude of light reflected or scattered from the volume    of the sample illuminated at said two or more incident angles;-   (iii) registering and stitching together in the Fourier Domain the    measurements from the two or more incident angles to create an    extended Fourier Field of measurements; and-   (iv) generating a three-dimensional image of an optical    characteristic of the sample by Fourier Transformation or digital    processing of the extended Fourier Field measurements.

According to a twelfth aspect of the present invention there is provideda method for performing high resolution optical imaging of a sample,said method comprising the steps of:

-   (i) illuminating, with a multi-wavelength optical beam, a volume of    a sample to be imaged in three spatial dimensions;-   (ii) sampling in the Fourier plane light reflected or scattered from    the illuminated volume of said sample;-   (iii) simultaneously capturing phase and amplitude information over    a range of wavelengths of the sampled reflected or scattered light;    and-   (iv) processing the phase and amplitude information to construct a    three-dimensional image of an optical characteristic of said sample    over said illuminated volume.

According to a thirteenth aspect of the present invention there isprovided an article of manufacture comprising a computer usable mediumhaving a computer readable program code configured to operate theapparatus according to any one of the first to sixth aspects, or toimplement the method according to any one of the seventh to twelfthaspects.

According to a fourteenth aspect of the present invention there isprovided an apertured reflector comprising: a total internal reflectionsurface for reflecting light; and one or more apertures that locallydisrupt the total internal reflection at said surface, for transmittinglight without reflection.

Preferably, the apertured reflector comprises two total internalreflection surfaces spaced apart by one or more localised index matchingregions that form the one or more apertures. More preferably, theapertured reflector comprises two prisms with polished optical surfacesthat form the two total internal reflection surfaces, fixedly attachedand spaced apart from each other with localised regions of an indexmatched adhesive that form the one or more apertures. The two totalinternal reflection surfaces are preferably spaced apart byapproximately 10 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described, by way ofexample only, with reference to the accompanying drawings in which:

FIG. 1 illustrates in schematic form the acquisition of B-scan data witha 2-D sensor array in a prior art line-field OCT system;

FIG. 2 illustrates a general scheme for mapping data from three spatialdimensions, equivalent to two lateral dimensions and one spectraldimension, onto a 2-D sensor array;

FIG. 3 illustrates an embodiment of the FIG. 2 scheme with sampling inthe Fourier plane;

FIG. 4 illustrates a spectral domain OCT apparatus configured forFourier plane sampling of light scattered or reflected from a sample,according to an embodiment of the present invention;

FIG. 5 illustrates another spectral domain OCT apparatus configured forFourier plane sampling of light scattered or reflected from a sample,according to an embodiment of the present invention;

FIG. 6 shows a scheme for reducing the loss of sample power that occurswhen analysing the polarisation of a reference beam and a returningsample beam;

FIG. 7 illustrates the mapping of a 2-D grid of beamlets dispersed ontoa 2-D sensor array;

FIG. 8 shows the magnitude of an exemplary 2-D Fourier spatial transformof an interferogram obtained with sampling in the Fourier plane;

FIG. 9 illustrates an embodiment of the FIG. 2 scheme with sampling inthe image plane;

FIG. 10 illustrates a spectral domain OCT apparatus configured for imageplane sampling of light scattered or reflected from a sample, accordingto an embodiment of the present invention;

FIG. 11 illustrates another spectral domain OCT apparatus configured forimage plane sampling of light scattered or reflected from a sample,according to an embodiment of the present invention;

FIG. 12 illustrates yet another spectral domain OCT apparatus configuredfor image plane sampling of light scattered or reflected from a sample,according to an embodiment of the present invention;

FIG. 13 illustrates a linear OCT apparatus configured for image planesampling of light scattered or reflected from a sample, according to anembodiment of the present invention;

FIG. 13A shows, for a given wavelength, a 2-D FFT of an interferogramobtained with the apparatus of FIG. 13;

FIG. 13B shows a 2D-FFT for dispersed wavelengths of an interferogramobtained with the apparatus of FIG. 13;

FIG. 14 illustrates a spectral domain OCT apparatus suitable forproviding angularly structured illumination to an ocular sample forenhanced lateral resolution and/or extended depth of focus, according toan embodiment of the present invention;

FIG. 14A shows in inset form a variation on the FIG. 14 apparatus, forproviding angularly structured illumination to a non-ocular sample; and

FIG. 15 shows an apertured reflector suitable for use in the apparatusof FIG. 14.

DETAILED DESCRIPTION OF THE INVENTION

It will be evident from the foregoing description of the prior art thatsingle shot acquisition of OCT data is advantageous not only forenhanced speed, especially for reducing motion artefacts with in vivosamples, but also for retaining phase coherence for digital refocusingor digital wavefront correction. Acquisition schemes for digitalreconstruction of the complex field that are not single shot, i.e. thatrequire multiple readouts of a sensor array, face the difficulty ofensuring phase registration between the data in each of the multipleframes. This difficulty is not insurmountable, but does requireadditional computation e.g. for stitching together single shot imagesacquired from adjacent sample volumes.

Existing spectrometer-based spectral domain OCT systems, such as thatdescribed in US 2014/0028974 A1, can acquire B-scans (one lateraldimension) in a single shot, but not single shot C-scans (two lateraldimensions). This is because one axis of the 2-D sensor array isoccupied by the wavelength dispersion, as shown in FIG. 1. Thislimitation can be overcome if the combined returning sample andreference wavefronts are sampled in the two lateral dimensions with asampling system that may for example comprise a 2-D lenslet array, aMEMS mirror array or a diffractive optical element (DOE), and theresulting sampling points dispersed onto separate sets of pixels of a2-D sensor array. The effect of this general scheme is to squeeze datafrom three spatial dimensions, equivalent to two lateral dimensions andone spectral dimension, onto a 2-D sensor array. The mapping ofdispersed sampling points onto separate sets of pixels can be ensured byappropriate orientation or positioning of the sampling points, e.g. thelenslets of a 2-D lenslet array, with respect to the wavelengthdispersive element. As shown schematically in FIG. 2, one particular wayof implementing this general scheme is to sample the combined wavefrontswith a 2-D lenslet array 10 comprising a rectilinear (X, Y) array oflenslets tilted at an angle θ with respect to the dispersive axis 11 ofthe dispersive element 4 that disperses the beamlets 14-1, 14-2 etc ontoa 2-D sensor array 6. Provided the tilt angle is chosen judiciously andthe sensor array has sufficiently fine pixels 12, as shown in a partialcutaway view, each beamlet 14-1, 14-2 etc from the lenslet array can bedispersed, e.g. by a grating 4, onto a unique set of pixels 16-1, 16-2etc of the sensor array, thereby enabling single shot C-scanacquisition.

Another way of expressing the general requirement for obtaining apreferred unique mapping is for the projection 13 of the sampledbeamlets onto the sensor array 6 to be suitably angled with respect tothe projection of the dispersive axis 11 of the dispersive element 4onto the sensor array. Other solutions, e.g. using 2-D lenslet arrayswith non-rectilinear arrangements of lenslets, will occur to thoseskilled in the art.

Ideally, the wavelength dispersive element 4 and sensor array 6 arearranged such that the projection of the dispersive axis 11 onto thesensor array is parallel to rows of pixels 12 in the sensor array, i.e.parallel to an axis of the sensor array as shown. In practice however,the dispersed images formed on the sensor array from each beamlet willgenerally have some degree of curvature such that the mapping, whileknown, is unlikely to correspond to single rows of pixels over anextended length.

The systems to be described below are generally designed to illuminate asmall contiguous area of a sample with a multi-wavelength collimated ornear-collimated optical beam of the order of 100 μm in diameter at thesample, and to capture an image of the interaction volume in a singlesnapshot with spatial resolution significantly better than the size ofthe illuminated area, e.g. around 3 μm or better. In preferredembodiments the contiguous illuminated area is kept relatively small,less than or equal to 500 μm×500 μm in area, more preferably less thanor equal to 200 μm×200 μm in area. This is generally necessitated by theavailable number of sampling points, i.e. the number of lenslets incommercially available lenslet arrays, but it also reduces the impact ofmultiple scattering that can severely degrade the resolution of fullfield, wavelength sequential apparatus. The phase coherence betweenscatterers in the sample enables accurate volume reconstruction withdigital correction of aberrations and an extended depth of focus. Largerlateral ranges can be achieved by scanning the illumination area, e.g.by laterally scanning the beam or the sample, and stitching togethersequentially captured volumes, preferably with adjacent volumespartially overlapping to facilitate accurate phase registration.Importantly, the simultaneous illumination of a contiguous area reducesthe sensitivity to crosstalk from multi-path scattering and to spuriousreflections from outside the coherence length.

In preferred embodiments the 3-D snapshots are captured with agrating-based spectral OCT system, in which a 2-D lenslet array samplesthe light reflected or scattered from a small contiguous illuminatedarea, and the resulting beamlets dispersed and imaged onto a 2-D sensor.Importantly, the resolution (number of pixels) of the sensor is muchlarger than the resolution of the lenslet array (number of lenslets),thus enabling both lateral and spectral information to be captured onthe 2-D sensor in a single snapshot. As described above regarding FIG.2, in preferred embodiments a 2-D rectilinear lenslet array is tiltedwith respect to the dispersive axis of the dispersive element to ensurethat each beamlet is dispersed onto a unique group of pixels. Asignificant advantage of grid-based sampling is that it enables the useof a simple imaging system, with no requirement for a high magnification‘microscope’ to match the sample illumination area to the 2-D sensor.Such a microscope would typically require a magnification of order 100,which demands complicated imaging optics. The reflected or scatteredfield can be sampled by the lenslet array in either the Fourier plane,i.e. the far field, which is a form of holoscopy, or in the image plane,i.e. the near field. For either case the lateral resolution isdetermined by the numerical aperture of the objective lens, and thelateral area captured in a single snapshot is determined by the productof the lateral resolution and the number of lenslets. Advantageously,sampling in the Fourier plane allows subsequent processing to recreatean imaging lens mathematically, with the possibility of varying thatlens for different parts of a sample.

Full range imaging can be achieved by mixing the signal with an off-axisreference beam to introduce a spatial carrier, enabling unambiguousphase measurement. Given a phase coherent signal, sampled over bothtransverse axes and wavelength, a number of well-known digitalrefocusing techniques can be applied. For example techniques developedfor swept source holoscopy can be applied to extend the depth of fieldor to compensate for aberrations.

We turn now to description of various 3-D spectral domain OCT systemsthat exploit the tilted lenslet array technique shown in FIG. 2 forsingle shot C-scan acquisition, e.g. over regions of up to 500 μm×500 μmin area and with lateral resolution of 3 μm or better. These systems arecapable of full range imaging, i.e. the ability to distinguish positiveand negative path length delays, wavefront correction such as digitalrefocusing and aberration correction, and enhanced resolution.Critically, the ability to capture the complex field in a singlesnapshot ensures that phase coherence is maintained throughout thesample volume, which is a requirement for accurate wavefront correction.

In certain embodiments the combined beams are sampled in the far field,i.e. in the Fourier plane. As illustrated schematically in FIG. 3, light18 reflected or scattered from a point (x′,y′) in a contiguousilluminated volume 19 of a sample 20 is collected with an objective lens22, mixed with an off-axis reference beam 24 and sampled in the Fourierplane with a 2-D lenslet array 10. In this configuration the sample 20,and preferably also the lenslet array 10, are approximately at a focalplane of the objective 22, recognising that a three-dimensional samplecannot be exactly at the focal plane throughout its entire depth. Afterpassing through an aperture array 25 the focused beamlets 14 arecollimated, dispersed and imaged onto a 2-D sensor array 6. The aperturearray 25 is optional, but serves to block scattered signals from outsidethe coherence length that would otherwise degrade the sensitivity of theapparatus. As explained previously, in preferred embodiments arectilinear 2-D lenslet array 10 is tilted with respect to thedispersive axis of the wavelength dispersive element to provide amapping of the dispersed beamlets onto unique sets of pixels 16-1, 16-2etc of the sensor array 6. Since the reflected or scattered signal 18 issampled in the Fourier plane, its lateral content is obtained from thespatial (lateral) frequency content of the sampled signal. The axialreflectivity profile of the interaction volume 19 is encoded in thespectral frequency content, as is usual in spectral domain OCT.Importantly, a spatial Fourier transform separates the positive andnegative components of the signal. A subsequent Fourier transform alongthe spectral axis 8 provides the full range reflectivity profile. Anumber of optical characteristics of the sample can be extracted withspatial resolution from this reflectivity profile, including for examplephase, reflectivity, refractive index, refractive index changes andattenuation. If the measurement system is polarisation sensitive, i.e.adapted to capture phase and amplitude information for at least firstand second polarisation states of the beamlets, then one or morepolarisation-related optical characteristics such as birefringence ordegree of polarisation can be extracted. Many if not all of theseoptical characteristics will generally be wavelength-dependent.

FIG. 4 shows a spectral domain OCT apparatus configured for Fourierplane sampling of light reflected or scattered from a sample 20, withhigh lateral resolution. In an illumination system of the apparatus,light from an optical fibre-coupled multi-wavelength or broadband source26 such as a superluminescent light emitting diode (SLED) is split witha 2×2 optical fibre coupler 28 into a sample arm 30 and a reference arm32. The splitting ratio of the 2×2 coupler may for example be 90/10sample/reference, or even 99/1, because in many practical applicationsthe reflectivity of the sample 20 will be low. The sample beam 34 iscollimated with a lens 36 then directed onto a sample 20 via aconverging lens 38 and an objective 22. In preferred embodiments theobjective has a relatively high numerical aperture to ensure highlateral spatial resolution. For example a 0.16 NA objective typicallyprovides a lateral spatial resolution of 3.0 μm. The purpose of theconverging lens 38 is to enable illumination of an extended contiguousvolume 19 of the sample, for example 100 μm in diameter. Since thesample 20 is at a focal plane of the objective 22, if this converginglens were omitted the illuminated region would be a diffraction-limitedspot rather than an extended region. It will be appreciated that asingle lens could be used in place of the collimating lens 36 and theconverging lens 38.

In a measurement system of the apparatus, reflected or scattered samplelight 18 from within the illuminated volume 19 is collected with theobjective 22 and directed to a beam splitter such as a beam-splittingcube 40 where it is mixed with an off-axis collimated reference beam 24.The combined beam is sampled in the Fourier plane with an appropriatelypositioned rectilinear 2-D lenslet array 10, optionally followed by anaperture array (not shown), and the resulting beamlets 14 are collimatedwith a lens 42, dispersed with a wavelength dispersive element in theform of a reflective grating 43, and focused via a lens 44 onto a 2-Dsensor array 6, from which the combined interferogram can be read out ina single frame for subsequent analysis by a processor 45 equipped withsuitable machine-readable program code. Alternatively, the dispersiveelement could be a transmissive grating or a prism. As described abovein relation to FIG. 2, the lenslet array 10 is preferably tilted withrespect to the dispersive axis of the grating 43 so that each beamlet ismapped onto a unique set of pixels of the sensor array 6. In oneparticular embodiment the lenslet array has 1000 lenslets in arectilinear 40×25 grid with a 300 μm pitch, and the 2-D sensor array isa 20 Megapixel CMOS camera with a pixel size of 5.5 μm.

The combined interferogram read out from the sensor array 6 represents awavelength-dependent measurement of a signal of light reflected orscattered from the interaction volume 19, where the signal is a functionof the phase and amplitude of the electric field vector of the reflectedor scattered light 18. Using mathematical techniques described below,these wavelength-dependent measurements can be processed to generatenumerical representations or construct a three-dimensional image of anoptical characteristic of the sample with spatial resolution over atleast a portion of the interaction volume 19. A number of opticalcharacteristics of the sample can be extracted, including for examplephase, reflectivity, refractive index, refractive index changes andattenuation. Many if not all of these optical characteristics willgenerally be wavelength-dependent. We note that the measurement systemcould be made polarisation sensitive, e.g. by inclusion of apolarisation walk-off element in front of the 2-D sensor array 6 asdescribed below with reference to FIG. 14. In this case one or moreoptical characteristics related to polarisation properties of thesample, such as birefringence or degree of polarisation, could also beextracted.

FIG. 5 shows another spectral domain OCT apparatus configured forFourier plane sampling of light reflected or scattered from a sample 20,with high lateral resolution. In this apparatus the sample beam 34 andreference beam 24 are generated and combined with a polarisation beamsplitter 41, quarter waveplates 46 and a polarisation analyser 48. Anadvantage of using a polarisation beam splitter and associatedpolarising optics instead of a power beam splitter 40 as shown in theFIG. 4 apparatus is that it avoids wasting 50% of the light from thebroadband source 26. As mentioned previously the reference beam 24 willgenerally be much more intense than the returning sample beam 18. Onemethod for compensating for this, as shown in FIG. 6, is to orient thepolarisation analyser such that its transmission axis 49 is close toparallel to the polarisation direction 50 of the low intensity returningsample beam, and therefore close to orthogonal to the polarisationdirection 52 of the much more intense reference beam. Compared to theusual practice of orienting the polarisation analyser at 45° to thepolarisation directions of both beams, this reduces the loss in samplepower from 3 dB to less than 1 dB. Returning to FIG. 5, betterequalisation of the reference and returning sample beam powers can alsobe achieved with an optional quarter waveplate 54 in the source arm,oriented such that the polarisation beam splitter 41 preferentiallydirects the source light 60 into the sample arm.

A combination of a converging lens 38 and a high NA objective 22 is usedto illuminate an extended contiguous volume 19 of a sample 20, forexample 100 μm in lateral diameter, similar to the case with theapparatus shown in FIG. 4. Reflected or scattered sample light 18 fromwithin the illuminated volume 19 is collected with the objective 22,mixed with the reference beam 24 and sampled in the Fourier plane withan appropriately positioned rectilinear 2-D lenslet array 10 followed byan optional aperture array (not shown). The beamlets 14 are collimatedwith a lens 42, dispersed with a wavelength dispersive element in theform of a transmissive grating 56, and focused via a lens 44 onto a 2-Dsensor array 6. Alternatively, the dispersive element could be areflective grating or a prism. As described above in relation to FIG. 2,the rectilinear lenslet array 10 is preferably tilted with respect tothe dispersive axis of the grating 56 so that each beamlet is mappedonto a unique set of pixels 16 of the sensor array 6. The combinedinterferogram can be read out from the 2-D sensor array in a singleframe for subsequent analysis by a processor 45 equipped with suitablemachine-readable program code. Again the interferogram represents awavelength-dependent measurement of a signal of light reflected orscattered from the illuminated volume 19, where the signal is a functionof the phase and amplitude of the electric field vector of the reflectedor scattered light 18. As before, these wavelength-dependentmeasurements can be processed to generate numerical representations orconstruct a three-dimensional image of an optical characteristic of thesample with spatial resolution over at least a portion of theinteraction volume 19.

It is generally preferable to interfere the returning sample beam with areference beam that is well collimated and covers all of the lenslets inthe array 10. This is straightforward in the FIG. 4 apparatus withappropriate selection of the reference arm collimating lens 58, but moredifficult in the FIG. 5 apparatus because the source beam 60 enteringthe beam splitter 41 is intentionally not collimated so as to illuminatean extended (not diffraction-limited) area of the sample 20. To this endthe reference arm of the FIG. 5 apparatus includes a NA convertor 62between the quarter waveplate 46 and the reference mirror 64 to converta smaller diameter divergent beam 66 into a larger diameter collimatedbeam 24. The NA convertor 62 comprises a larger diameter lens 70 and asmaller diameter lens 72 (such as a lenslet) separated by a distanceequal to the focal length of the larger lens, f₁. These two lenses, incombination with the reference mirror 64, bring the divergent beam 66 toa focus 73 inside the lenslet 72. Consequently the lenslet has norefractive power on the return path, so that the outgoing beam 74 withincreased NA is collimated by the larger lens 70.

We turn now to a description of an analysis of interferometric dataobtained when sampling in the Fourier plane. With Fourier planesampling, every beamlet 14 contains phase and amplitude information fromevery point in the interaction volume 19, but at different discreteangles. Spatial information is therefore encoded as angular information.

For simplicity we consider the scattering or reflection from a singlepoint at position (x′,y′) as shown in FIG. 3, and with depth Δz.Assuming that the scattering or reflection point is close to the focalplane of the objective lens 22, the collimated field incident upon thelenslet array 10 will be a plane wave with an incident angle x′/f. Theinterferometric signal incident on the lenslet array at position X, Ycan thus be expressed as:

$\begin{matrix}{{I\left( {X,Y,x^{\prime},y^{\prime}} \right)} = {{S(k)}{R\left( {x^{\prime},y^{\prime}} \right)}^{1\text{/}2}\mspace{14mu} {\cos \left( {k\left( {{\Delta \; z} + \frac{X\left( {x^{\prime} - x_{0}} \right)}{f} + \frac{Y\left( {y^{\prime} - y_{0}} \right)}{f}} \right)} \right)}}} & (1)\end{matrix}$

where R(x′,y′) is the sample reflectivity, S(k) is the spectral powerdistribution, f is the focal length of the objective lens 22, and x₀ andy₀ are related to the angle of the reference beam 24 with respect to theaxis of the objective lens (or to the axis of the lenslet array 10).

To first order, the interferometric signal component at the aperturearray 25 for the lenslets (of circular aperture) can be approximated by:

$\begin{matrix}{{I\left( {X_{i},Y_{j},x^{\prime},y^{\prime}} \right)} = {{{S(k)}{R\left( {x^{\prime},y^{\prime}} \right)}^{1\text{/}2}\mspace{14mu} \left( {{\cos \left( {k\left( {{\Delta \; z} + \frac{X\left( {x^{\prime} - x_{0}} \right)}{f} + \frac{Y\left( {y^{\prime} - y_{0}} \right)}{f}} \right)} \right)} \otimes {{circ}\left( {X,Y,D} \right)}} \right)}_{{X = X_{i}},{Y = Y_{j}}}}} & (2)\end{matrix}$

where X_(i), Y_(j) describe the axis of the lenslet, D is the pitch ofthe lenslet array, and circ(X,Y,D)=1 for X²−Y²<(D/2)² and 0 otherwise.

From the combined interferogram measured by the 2-D sensor array 6 andknowledge of the wavelength mapping for each lenslet onto the 2-D sensorarray we can extract a set of interferograms I_(i,j)(k_(l)) where i, jdenote the lenslet positions within the lenslet array 10 and k_(l)denotes the wavenumbers resolved by the spectrometer (i.e. the grating)as illustrated in FIG. 7. It is convenient to consider theinterferograms I_(i,j)(k_(l)) as a sequence of two dimensionalinterferograms, one for each of M distinct wavenumbers. The dimension ofeach 2-D interferogram is equal to that of the lenslet array, forexample 25 rows×40 columns. As such, the analysis is analogous to thatof full-field swept source holoscopy (Hillmann et al, Optics Express20(19), 21247-21263 (2012)), the key difference being that the lowsampling resolution of the lenslet array compared to that of aphotodetector array (e.g. 300 μm lenslet pitch compared to 5 μm pixels)limits the field of view achievable in a single snapshot. As the sampleis measured in the Fourier plane, the image plane is obtained byapplying a 2-D spatial Fourier transform to each interferogram.Advantageously, with an off-axis reference the Fourier transform can beused to separate positive and negative spatial frequency components ofthe interferogram, so that a subsequent 1-D FFT along the spectral axisof the positive spatial frequency component achieves full axial depthrange. This can be readily seen from the lateral Fourier components ofthe cosine term in equation (1):

$\begin{matrix}{{\delta \left( {k_{x} \pm \frac{k\left( {x^{\prime} - x_{0}} \right)}{f}} \right)}{\delta \left( {k_{y} \pm \frac{k\left( {y^{\prime} - y_{0}} \right)}{f}} \right)}e^{{\square\; i}\; 2k\; \Delta \; {z{({x^{\prime},y^{\prime}})}}}} & (3)\end{matrix}$

The phase of the respective terms is now dependent on the sign of Δz.

We note that if the sample is on one side only of the zero delay, anoff-axis reference is not required. The complex signal with unambiguousphase is obtained by a first 1-D FFT along the spectral axis, and thenfor positive delays, a subsequent spatial 2-D FFT. So for a givenlateral bandwidth the lateral range is doubled compared to a full rangedsystem.

As an illustration of the field of view achievable with the Fourierplane sampling spectral OCT apparatus shown in FIG. 4 or 5, we considerthe following set of parameters. The lenslet array 10 has pitch P=300μm, NA=0.08, and spot size=0.61 λ/NA=6.1 μm at wavelength λ=0.8 μm. Forthe objective lens 22 we assume f=40 mm and diameter D=11 mm (NA=0.14),giving an expected resolution of 3.6 μm. The maximum lateral range(Δx=x−x₀) for a full range system can be estimated from the Nyquistlimit (λ·f)/Δx)>2P, which gives Δx<55 μm. The lateral range for cases inwhich delays are on one side only of the zero delay is twice this value,Δx<110 μm. Here we assume unit magnification between the objective lens22 and the lenslet array 10, and note that a lateral field of 55 μmgives a change in the focal position of the lenslet array of <0.5 timesthe focal spot size of each lenslet.

FIG. 8 illustrates the magnitude of the 2-D spatial Fourier transform offor the above parameters, with the reference beam offset to ensure thatthe positive and negative frequency components 76, 78 are separated. Inthis particular example the reference beam is offset in both thehorizontal and vertical axes, and we have subtracted out thenon-interferometric terms. Either the positive frequency component 76 orthe negative frequency component 78 can be extracted by filtering, andthe frequency offset removed. From equation (3) we see that for a givensample position the spatial frequency is wavelength dependent. Thiswavelength dependence is removed by multiplication with a wavelengthdependent phase factor prior to taking the Fourier transform over thespectral components, to obtain the full range depth profile.

In other embodiments the combined beams are sampled in the image plane.As illustrated schematically in FIG. 9, light 18 reflected or scatteredfrom a point (x′,y′) in an illuminated volume 19 of a sample 20 ismagnified and re-imaged onto a lenslet array 10. The complex field isobtained by mixing the returning sample light 18 with an off-axisreference beam 24 having an angle of incidence a. As in Fourier planesampling, the focused beamlets 14 from the lenslet array are passedthrough an optional aperture array 25 then collimated, dispersed andfocused onto a 2-D sensor array 6. In this case the reference beam 24 isrequired to be off-axis to separate the positive and negative frequencycomponents of the signal. As explained previously, in preferredembodiments the lenslet array is rectilinear in configuration and tiltedwith respect to the dispersive axis of the dispersive element to providea mapping of the dispersed beamlets onto unique sets of pixels 16-1,16-2 etc of the sensor array 6. The complex field is obtained from afirst spatial Fourier transform across the sampled field, settingnegative frequency components to zero and then applying an inverseFourier transform. As in the Fourier plane case a subsequent Fouriertransform along the spectral axis 8 provides the full-range reflectivityprofile of the illuminated volume 19 of the sample 20. A number ofoptical characteristics of the sample can be extracted with spatialresolution from this reflectivity profile, including for example phase,reflectivity, refractive index, refractive index changes andattenuation, as well as birefringence and degree of polarisation if themeasurement system is polarisation sensitive.

FIG. 10 shows a spectral domain OCT apparatus configured for image planesampling of light reflected or scattered from a sample 20, with highlateral resolution. In an illumination system of the apparatus, lightfrom an optical fibre-coupled multi-wavelength or broadband source 26 issplit with a 2×2 optical fibre coupler 28 into a sample arm 30 and areference arm 32. As with the FIG. 4 apparatus the splitting ratio ofthe 2×2 coupler may for example be 90/10 or 99/1 sample/reference. Thesample beam 34 is collimated with a lens 36 then directed onto anextended contiguous volume 19 of a sample 20 via a converging lens 38and an objective 22. In preferred embodiments the objective has arelatively high numerical aperture to ensure high lateral spatialresolution. For example a 0.16 NA objective typically provides a spatialresolution of 3.0 μm.

In a measurement system of the apparatus, reflected or scattered samplelight 18 from within the interaction volume 19 is collected with theobjective 22 and directed to a beam splitter such as a beam-splittingcube 40 where it is mixed with an off-axis collimated reference beam 24.The combined beam is sampled in the image plane with an appropriatelypositioned 2-D rectilinear lenslet array 10, optionally followed by anaperture array (not shown), and each beamlet 14 is collimated with alens 42, dispersed with a wavelength dispersive element in the form of areflective grating 43, and focused via a lens 44 onto a 2-D sensor array6, from which the combined interferogram can be read out in a singleframe for subsequent analysis by a processor 45 equipped with suitablemachine-readable program code. Alternatively, the dispersive elementcould be a transmissive grating or a prism. As described above inrelation to FIG. 2, the lenslet array 10 is preferably tilted withrespect to the dispersive axis of the grating 43 so that each beamlet 14is mapped onto a unique set of pixels of the sensor array 6. In oneparticular embodiment the lenslet array has 1000 lenslets in arectilinear 40×25 grid with a 300 μm pitch, and the 2-D sensor array isa 20 Megapixel CMOS camera with a pixel size of 5.5 μm. With this imageplane sampling scheme, each beamlet 14 contains phase and amplitudeinformation from a different portion of the interaction volume 19.

As before, the combined interferogram read out from the sensor array 6represents a wavelength-dependent measurement of a signal of lightreflected or scattered from the interaction volume 19, where the signalis a function of the phase and amplitude of the electric field vector ofthe reflected or scattered light 18. Using mathematical techniquesdescribed below, these wavelength-dependent measurements can beprocessed to generate numerical representations or construct athree-dimensional image of an optical characteristic of the sample withspatial resolution over at least a portion of the interaction volume. Wenote that the measurement system could be made polarisation sensitive,e.g. by inclusion of a polarisation walk-off element in front of the 2-Dsensor array 6 as described below with reference to FIG. 14. In thiscase the optical characteristic could be related to a polarisationproperty of the sample, such as birefringence or degree of polarisation.

FIG. 11 shows another spectral domain OCT apparatus configured for imageplane sampling of light reflected or scattered from a sample 20, withhigh lateral resolution. In this apparatus the sample beam 34 andreference beam 24 are generated and combined with a polarisation beamsplitter 41, quarter waveplates 46 and a polarisation analyser 48. Asdiscussed above regarding FIG. 6, the polarisation analyser 48 can beoriented such that its transmission axis is close to parallel to thepolarisation direction of the low intensity returning sample beam, toreduce the loss in sample power. Better equalisation of the referenceand returning sample beam powers can also be achieved with an optionalquarter waveplate 54 in the source arm, oriented such that thepolarisation beam splitter 41 preferentially directs the light from thebroadband source 26 into the sample arm.

A combination of a converging lens 38 and a high NA objective 22 is usedto illuminate an extended contiguous volume 19 of a sample 20, forexample 100 μm in lateral diameter, similar to the case with theapparatus shown in FIG. 10. Reflected or scattered sample light fromwithin the illuminated volume 19 is collected with the objective 22 andmixed with the reference beam 24, noting that the reference mirror 64 isangled in the non-dispersive axis so that the reference beam is off-axiswith respect to the returning sample beam. The combined beam is sampledin the image plane with an appropriately positioned 2-D rectilinearlenslet array 10, followed by an optional aperture array (not shown),and the resulting beamlets 14 collimated with a lens 42, dispersed witha wavelength dispersive element in the form of a transmissive grating56, and focused via a lens 44 onto a 2-D sensor array 6. Alternatively,the dispersive element could be a reflective grating or a prism. Asdescribed above in relation to FIG. 2, the lenslet array 10 ispreferably tilted with respect to the dispersive axis of the grating 56so that each beamlet is mapped onto a unique set of pixels 16 of thesensor array 6. The combined interferogram can be read out from the 2-Dsensor array in a single frame for subsequent analysis by a processor 45equipped with suitable machine-readable program code. As with the FIG.10 apparatus, each beamlet 14 contains phase and amplitude informationfrom a different portion of the illuminated volume 19.

FIG. 12 shows yet another spectral domain OCT apparatus configured forimage plane sampling, in this case of light reflected or scattered froma 2-D grid of discrete spots 79 on a sample 20 rather than from anextended contiguous area. In an illumination system of this apparatus, a2-D lenslet array 81 in the sample arm separates the sample beam into agrid of beamlets 80 that are focused onto the sample via lenses 82 and84 and a mirror 86. Optionally this mirror can be scanned in one or twoaxes to translate the focused beamlets across the sample, e.g. toanalyse different regions or fill in the gaps between the beamlets.Alternatively the sample 20 can be mounted on a translation stage. Thelenses 82 and 84 generally form a high magnification system, e.g. 100×,and it may be preferable to include additional lenses to perform themagnification in two or more stages.

In a measurement system of the apparatus, sample light reflected orscattered from the discrete spots 79 is collimated by the sample armlenslet array 81, relayed to the combined arm lenslet array 10 by meansof a 4F lens system 88, and mixed with a reference beam 24 renderedoff-axis by angling the reference mirror 64 in the non-dispersive axis.As in the FIG. 11 apparatus the combined beam passes through apolarisation analyser 48, then is sampled in the image plane with anappropriately positioned lenslet array 10 followed by an optionalaperture array (not shown). The resulting beamlets 14 are collimatedwith a lens 42, dispersed with a wavelength dispersive element in theform of a transmissive grating 56, and focused via a lens 44 onto a 2-Dsensor array 6 to form a combined interferogram. Alternatively, thedispersive element could be a reflective grating or a prism. Thecombined interferogram can then be read out from the 2-D sensor array ina single frame for subsequent analysis by a processor 45 equipped withsuitable machine-readable program code. The combined interferogramrepresents a wavelength-dependent measurement of a signal of lightreflected or scattered from the grid of discrete spots 79, where thesignal is a function of the phase and amplitude of the electric fieldvector of the reflected or scattered light.

Scanning of the mirror 86 or translation of the sample 20, if enabled,can be controlled conveniently in synchronisation with read out of thesensor array by means of the processor 45 when equipped with suitablemachine-readable program code.

We turn now to a description of an analysis of interferometric dataobtained when sampling in the image plane light scattered or reflectedfrom an extended contiguous volume 19 as shown in FIGS. 10 and 11.Assuming that the sample 20 is in a focal plane of the objective lens 22as shown in FIG. 9, the interferometric signal component incident atposition X, Y upon the lenslet array 10 can be expressed as:

$\begin{matrix}{{I\left( {X,Y,k} \right)} = {{S(k)}{R\left( {\frac{X}{M},\frac{Y}{M}} \right)}^{1\text{/}2}\mspace{14mu} {\cos \left( {k\left( {{2\Delta \; z} + {Y\mspace{14mu} {\sin (\alpha)}}} \right)} \right)}}} & (4)\end{matrix}$

where M is the magnification of the lens system 22, 38 in the sample armand a is the incident angle of the reference beam 24 at the lensletarray 10 as shown in FIG. 9. In this case the reference beam is assumedto be aligned with the Y-axis of the lenslet array. Importantly, forfull range imaging a must be large enough to separate the positive andnegative frequency components of the interferometric signal.

The analysis follows an analogous approach to that used in off-axisswept wavelength OCT, described for example in Huang et al, AppliedOptics 52(5), 958-965 (2013), or in Fechtig et al, Journal of ModernOptics 2014 (DOI: 10.1080/09500340.2014.990938). The complex field isobtained by taking the Fourier transform along the Y axis, removing thenegative frequency components and the frequency offset, and thenapplying an inverse Fourier transform to obtain the complexinterferogram I_(i,j)(k) where i, j denote a lenslet at position (X_(i),Y_(j)).

We assume similar experimental parameters to the previous far-fieldcase, i.e. a resolution of 3.6 μm (objective NA=0.14), a transverserange of 55 μm and lenslet pitch P=300 μm. A large magnification betweenthe sample and the lenslet array transforms the high numerical aperturehigh resolution sample information to a lower numerical aperture spot ofdimensions comparable to the lenslets that can optimally interfere withthe reference beam after they are both focused by the lenslet array.This range of incident angles is approximated by Δθ<λ/(2P) i.e. ˜½ theAiry radius. A magnification of NA/Δθ≈100 will therefore ensure thatrays emitted from the sample are captured. We note that this isequivalent to requiring the lenslet array pitch to be smaller than themagnified resolvable spot size.

In the above discussion it was assumed that the sample was in the focalplane of the objective lens. In general however, with three-dimensionalsamples the majority of the interaction volume will be somewhatdisplaced from the focal plane. Scattering from points away from thefocal plane gives rise to curved wavefronts at the lenslet array. Thecapture of partially overlapping consecutive 3-D snapshot samplesenables accurate phase registration of datasets, to which digitalrefocusing techniques can be applied using a processor equipped withsuitable machine-readable program code, either before or after thesnapshot samples are stitched together to form a 3-D composite image.Alternatively, digital refocusing can be applied directly to lateralpoints at the centre of the snapshot datasets, so as to avoid refocusingstitched datasets. Digital refocusing requires a first measurement ofthe signal with unambiguous phase and thus can be applied to both a fullrange system with an off-axis lateral reference and a system with alldelays of the same sign and an on-axis lateral reference. The signal canbe digitally refocused by adapting one of a number of well knowntechniques described for full field and line field systems, for examplein Kumar et al, Optics Express 22(13), 16061-16078 (2014), or modelssuch as that used in Fechtig et al, Journal of Modern Optics 2014 (DOI:10.1080/09500340.2014.990938). Although these approaches are applied toOCT systems that sample the image plane, they can be adapted to samplingof the Fourier plane. The digital focusing technique described inHillmann et al, Optics Express 20(19), 21247-21263 (2012) can bedirectly applied to the case in which we sample in the Fourier plane.

As an alternative embodiment to the various spectral domain OCTapparatus described previously, FIG. 13 shows a linear OCT apparatusconfigured for image plane sampling of light scattered or reflected froma sample. In an illumination system of the apparatus, light from amulti-wavelength or broadband optical source 26 is split with a beamsplitter 40-1 to form a sample beam 34 and a reference beam 24. As inthe apparatus shown in FIG. 10, an extended contiguous volume region 19of a sample 20 is illuminated by a collimated sample beam 34. In ameasurement system of the apparatus reflected or scattered light 18 iscollected and magnified with a telescope system comprising lenses 22 and38, and sampled in the image plane by an appropriately positionedrectilinear 2-D lenslet array 10 followed by an optional aperture array(not shown). The resulting beamlets 14 are then collimated with a lens42 and directed onto a 2-D sensor array 6 via another beam splitter40-2. In contrast to the previously described spectral domain approach,a collimated reference beam 24 is dispersed, e.g. with a reflectivegrating 43, then mixed with the sample field directly onto the sensorarray 6. The rectilinear lenslet array 10 is preferably tilted withrespect to the dispersive axis of the grating 43 so that thewavelength-dependent spatial frequency components obtained from a 2-DFFT for each beamlet 14 are mapped onto a unique set of pixels of the2-D sensor array 6. These wavelength-dependent spatial frequencycomponents can be read out from the 2-D sensor array for subsequentanalysis by a processor 45 equipped with suitable machine-readableprogram code. Each component represents a wavelength-dependentmeasurement of a signal of light reflected or scattered from a differentportion of the interaction volume 19, where the signal is a function ofthe phase and amplitude of the electric field vector of the reflected orscattered light 18.

For a given wavelength, a 2-D FFT 134 of the corresponding interferogramread out from the sensor array 6 is illustrated in FIG. 13A. Eachbeamlet corresponding to a lenslet in the lenslet array 10 isrepresented by a distinct spatial frequency component 136 with bothamplitude and phase. The dispersive grating 43 in the reference armenables the amplitude and phase of each wavelength component of eachbeamlet to be measured. Because the lenslet array 10 is angled withrespect to the dispersive axis of the grating 43 the spectral content ofthe beamlets remain distinct in the frequency domain, allowing us toobtain a distinct 2D-FFT 138 for each beamlet as shown in FIG. 13B. A1-D Fourier transform over the spectral component of the positivespatial frequency content provides the full range 3-D depth profile. Forsimplicity the dispersed beamlets 140 are depicted in FIG. 13B as beingparallel, although in practice their slopes will generally vary with theincident angle of the beamlets 14 onto the sensor array 6 and hence willnot be identically parallel. Nevertheless, with appropriate systemdesign the variations in slope between the dispersed beamlets 140 willbe sufficiently small such that they do not overlap in spectral content.

An advantage of this approach compared to the spectral domain OCTapproach is that it potentially avoids an expensive and difficult toalign spectrometer. As with the spectral domain approach, a referencebeam tilted in the axis perpendicular to the dispersive axis allows thepositive and negative frequency terms 142 and 144 to be separated, asseen in FIG. 13A. Consequently the 1-D FFT along the spectral axisenables a full-range axial measurement. Similarly, in analogy with thespectral domain approach, digital wavefront correction and measurementcan be implemented. Linear OCT however suffers from the well-known timedomain sensitivity penalty compared with spectral domain OCT. Forapplications in which a comb of discrete wavelengths can be used, i.e.sparse axial reflectivity profiles, this sensitivity penalty can bereduced.

As mentioned previously, there is a trade-off in OCT imaging betweentransverse resolution and depth of field. Fundamentally, this trade-offarises because higher NA lenses enable smaller spot sizes, and thereforeincreased transverse resolution, but at the cost of reduced depth offield.

FIG. 14 shows an apparatus for retinal imaging, in which the retina 89of an eye 90 is illuminated at a number of different angles through alensing system with a limited numerical aperture. For ocular samples thenumerical aperture is limited by the size of the pupil 92, while inother microscopic optical systems or for other samples the numericalaperture of the imaging system may be limited by the size of the opticalelements. Importantly, the angularly structured illumination provided bythis apparatus, i.e. the illumination of a volume 114 of the retina attwo or more incident angles, enables higher lateral resolution thanwould otherwise be possible with an imaging system of limited numericalaperture, while retaining the increased depth of field advantage oflower numerical aperture. Note that this angularly structuredillumination is distinct from the different angles incident onto thecornea 94, controlled by the mirror 112, required to illuminatedifferent volumes of the retina 89 as explained below.

Light from a superluminescent light emitting diode (SLED) 26 or someother broadband or multi-wavelength source is used both to probe theretina 89 and to measure interferometrically properties of the retinavia reflected or scattered light. In most general form, the light source26 should emit light having at least first and second wavelengths. Whenusing polarisation-sensitive detection as described below, the lightsource should be polarised, i.e. emits light of a given polarisationstate. In an illumination system of the apparatus, the SLED output isformed into a beam by a collimating element 36 and split into a samplepath 30 and a reference path 32 by a polarisation-insensitive beamsplitting element 40 such as a conventional polarisation independentbeam-splitting cube as used for example in the FIG. 4 apparatus. Thesample path beam is reduced in size with a beam reducer 96 such as areversed Gaussian beam expander and directed to a beam steering element98 such as a two-axis MEMS mirror. This beam steering element is adaptedto direct the sample path beam in a number of paths that become paralleland spatially separated after traversing a parallelising element 100. Inthe illustrated embodiment this parallelising element is a lenspositioned one focal length away from the MEMS mirror 98, although inother embodiments it could be a prism or a mirror. In certainembodiments the parallelising element also resizes the sample beam. Eachresultant beamlet 101 should be significantly smaller in diameter thanthe pupil 92 of the sample eye, so that it will illuminate a small areaof the retina 89, preferably with a diameter in the range of 50 to 500μm in the present case. For a normal relaxed eye a parallel set ofbeamlets 101 will come to illuminate the same point of the retina,though for a highly myopic eye it may be necessary to adjust theposition of the parallelising element 100 to provide a non-parallel setof beamlets that will largely overlap at the retina. In an alternativeembodiment the beamlets 101 are generated simultaneously with adiffractive optical element (DOE) instead of sequentially with the MEMSmirror 98, in which case the illumination onto the retina will be in theform of a simultaneous angularly structured illumination. Generallyspeaking the sequential embodiment is more straightforward analytically,so long as the analysis is not unduly influenced by sample movementbetween frames.

The sequence or simultaneous array of beamlets 101 is passed through abeam splitting element, which in preferred embodiments comprises anapertured reflector 102, wherein they are able to pass through a numberof discrete apertures 104 without significant loss, at positions thatcan be addressed by different angles of the MEMS mirror 98 (forsequential beamlets) or the structure of a DOE (for simultaneousbeamlets). In general form the apertured reflector has a surface,preferably a total internal reflection surface 110, for reflectinglight, and one or more apertures 104 that locally disrupt the totalinternal reflection at that surface, for transmitting light withoutreflection. In one embodiment the apertured reflector comprises a prismwith a polished optical surface for total internal reflection, and oneor more apertures in the form of drilled holes that disrupt the totalinternal reflection. In a preferred embodiment illustrated in FIG. 15the apertured reflector 102 comprises a pair of prisms 106 that providetotal internal reflection 107 at one or other of the polished opticalsurfaces 110 except in the apertures 104 defined by droplets 108 of anindex matched adhesive that fixedly attach and space apart thereflective surfaces. The separation between the prisms 106 imparted bythe droplets of adhesive is not a critical parameter, but may forexample be in the range of 5 to 500 μm, e.g. approximately 10 μm.Preferably the prisms 106 are right angle prisms as shown. The choice ofan apertured beam splitting element 102 may be particularly advantageousover a conventional power beam splitter as it enables the illuminationof a sample and capture of back-reflected light independent ofpolarisation and with low intrinsic power losses. Obviously some signallight will be lost through the apertures 104 in the return path, butthis can be minimal when the illuminating beamlets 101 are small becausethe overall area of each reflective surface 110 is significantly largerthan the apertures 104. The beam splitting element 102, in whateverform, is preferably polarisation independent, and any residualpolarisation sensitivity can be calibrated out if necessary.

Returning to FIG. 14, the beamlets passing through the aperturedreflector 102 are then incident onto an angularly variable element inthe form of a steerable mirror 112, optionally in combination with anoptical relay system (not shown), which directs them onto a commoninteraction volume 114 of the retina 89. It will be appreciated that thebeamlets 101 are directed onto the common interaction volume 114 atdifferent incident angles, determined by the beam steering element 98and the optical power of the eye 90. The position of the commoninteraction volume 114 on the retina can be varied, e.g. to position114′, by angular adjustment of the mirror 112 so that a larger area ofthe retina can be imaged via collection of a number of images that canbe stitched together to create a composite image. Light 18 scattered orreflected from the interaction volume 114 for a given position of themirror 112 is then captured across the whole pupil 92, collimatedroughly by the eye's optical power then directed by the mirror 112 tohave an equivalent propagation direction independent of the point ofillumination of the retina for the case of an ideal relaxed eye focusedat infinity. After being directed away from the optical source 26 by theapertured reflector 102, the roughly collimated return beams 116 arefocused down by a lens 118 to form an image of the retina. Preferablythe lens 118 is of variable focus to provide some gross aberrationcorrection to adapt for the large variations of myopia and astigmatismtypically found in a clinical setting. Variable focus lenses areprovided for example by Varioptic.

We note that the apparatus shown in FIG. 14, unlike the FIG. 4 apparatusfor example, does not require an objective lens 22 to illuminate thecommon interaction volume 114 and collect the scattered or reflectedlight 18, because these functions are performed by the cornea and lensof the sample eye 90. As illustrated in FIG. 14A, the sample-relatedportion of the FIG. 14 apparatus can be adapted for providing angularlystructured illumination to other types of sample 20 by the inclusion ofan objective lens 22 or other optical power element, with the samplelocated at or close to a focal plane of this lens. The objective lensdirects the beamlets 101 onto a common interaction volume 114 of thesample 20, and collects the scattered or reflected light 18. In place ofthe angularly variable mirror 112, an X, Y translation stage 119 can beused to move the sample for imaging further volumes 114′. It will beappreciated that there are many other schemes for providing angularlystructured illumination to one or more volumes of a non-ocular sampleand collecting the scattered or reflected signal light, involving forexample back illumination, beam splitters or combiners, and rotation ofthe sample.

The image of the retina is passed through a beam combiner 120 into ameasurement system of the apparatus, which includes an interferometer.In the illustrated embodiment the beam combiner 120 is an aperturedreflector similar to the element 102, but with a single aperture 104 forpassing the image of the retina. This allows the returning sample beamto be combined with the reference beam 24 and sampled in the Fourierplane with an appropriately positioned 2-D lenslet array 10.

We turn now to description of the path of the reference beam 24, whichis reflected at the beam splitter 40 and passed through a delay linecomprising a pair of right angle prisms 106A, 106B that approximatelymaps the group delay of the sample arm light travelling to and from thesample eye 90. This delay line may incorporate a dispersion equalisationelement 124 to ensure that dispersion in the reference arm 32 is similarto that in the sample arm 30. Optionally, the reference arm can includea polarisation modifying element 126 such as a half wave plate or apolariser to create a given polarisation state for the reference beam.Following the delay line, the reference beam 24 is focused onto a totalinternal reflection surface 110 of the apertured reflector 120 to form afocal point near to the position of the image of the retina (i.e. nearto the aperture 104). The reflected reference beam and the transmittedsample beam are then approximately collimated by a lens 122 thatconverts the far field angular distribution into a spatial distribution,which can be sampled over a plane 2-D surface with a lenslet array 10. Aco-registered aperture array 25 is preferably included to reject straylight that would compromise the resolution of the 2-D dispersive opticalsystem 128 described below.

The use of an apertured reflector for the beam combiner 120 isparticularly advantageous in this configuration as both the sample andreference beams, being focused at the beam combiner 120, can be passedinto the interferometer portion of the apparatus without significantloss. If the lateral displacement between the sample and reference focalpoints is small then the impact of the offset on the fringe contrast inthe interferometer can be minimised. Consequently the apparatus is ableto provide a very high signal to noise ratio for a given illuminationpower on the sample, which obviously must be limited for ocular samples.

The 2-D dispersive optical system 128 comprises a first collimating lens42 positioned about one focal length away from the aperture array 25, awavelength dispersive element in the form of a transmissive grating 56providing dispersion along one axis, and a second lens 44 for focusingthe dispersed array of grid points onto a 2-D sensor array 6 such as aCMOS camera or other focal plane array. In alternative embodiments thedispersive element could be a reflective grating or a prism. Note thatfor simplicity of illustration, representative ray paths through thedispersive engine 128 are not shown in FIG. 14. As before, theorientations of the lenslet array 10 and the dispersive axis of thegrating 56 are chosen so that the spectral dispersive lines created fromeach of the beamlets emerging from the apertures are slightly offsetlaterally as shown in FIG. 2. A polarisation walk-off element 130 suchas a YVO₄ plate is provided to split the polarisation state of both thesignal and reference beamlets, providing two wavelength-dispersed lines132 for each sampling point (aperture or lenslet) as shown. Inalternative embodiments without polarisation-sensitive detection thewalk-off element 130 is omitted.

The operation of the imaging apparatus shown in FIG. 14 is firstlydescribed for its normal lateral resolution mode, i.e. without angularlystructured illumination, which is limited by the NA of the sample eye90. During a single frame acquisition period of the CMOS camera 6,preferably operated in global shutter mode, the SLED 26 is pulsed for aperiod of time short enough to avoid significant motion artefacts in theeye. The sample beam is directed through a single aperture 104 of theapertured reflector 102 to illuminate a volume 114 of the retina 89through a specific region of the pupil 92. The dispersed interferometricimage of the back-reflected or scattered light from the illuminatedvolume, with or without the polarisation splitting conferred by thewalk-off element 130, is then read digitally from the CMOS camera 6 andprocessed with a processor 45 equipped with suitable machine readableprogram code to provide a pixel-to-wavelength map for each of thesampling points defined by the lenslet array 10. The wavelengths can beconverted into a linearised k-vector through interpolation as is wellunderstood in the field of spectral domain OCT to construct a phase andamplitude map across the surface of the illuminated volume 114 for aregularly spaced set of k-vectors. As the set of sample pointscorresponds to a sampling of the electric field vector in the Fourierplane it is possible to use a Fourier transform to construct or generatea 3-D image or representation of the illuminated sample volume 114. Useof digital aberration correction and/or digital refocusing may beapplied to maintain the lateral resolution of the image across anenhanced depth of field, compared to that which could be achieved in thecase of a single scanning beam with an equivalent numerical aperturelimitation.

The 3-D image or representation constructed or generated may be of asingle polarisation amplitude or phase measurement of the reflected orscattered light, suitable for extraction of an optical characteristic ofthe sample such as phase, reflectivity, refractive index, refractiveindex changes or attenuation. Alternatively, if the detection system ispolarisation-sensitive e.g. by virtue of a polarisation walk-off element130, the image or representation may be of a polarisation property ofthe sample, such as birefringence or degree of polarisation, which maybe indicative of the type of material being sampled. These measurementtechniques are well understood for OCT systems (e.g. scanning, timedomain or full field OCT systems) and can now be applied in astraightforward fashion.

In preferred embodiments the polarisation-sensitive detection system iscomplemented with a polarisation control element 134 such as avoltage-controlled liquid crystal element in the sample arm, to enableillumination of the sample volume 114 with light of a second, differentpolarisation state. In this case the measurement system makes a second,additional set of simultaneous measurements of the reflected orscattered light 18, and the processor 45 processes both sets ofmeasurements to construct or generate a three-dimensional image orrepresentation of one of more polarisation properties of the sample.This modification to the apparatus avoids, for example, the situation ofbeing unable to measure sample birefringence if it happens to beparallel to an input polarisation state. In general, while illuminationwith a single polarisation and subsequent polarisation-sensitivedetection can often provide a clinically useful contrast mechanism, theability to make two or more separate measurements of a sample withdifferently polarised illumination states allows one to obtain a morecomplete description or representation of the polarisation properties ofthe sample.

To image an additional portion of the retina 89 the angular position ofthe mirror 112 is adjusted to illuminate a second volume 114′, which ispreferably adjacent to the first volume 114 and with a small overlap tofacilitate registration of the composite stitched image. The process canbe repeated for a number of angles of the mirror 112 to construct athree-dimensional composite image of an increasingly large area of theretina. Each of the individual images can be thought of as a numericalrepresentation of an optical characteristic of the retina over therespective volume. Of particular interest is the fact that digitalrefocusing and/or aberration correction can be carried out for each ofthe individual volume images so that off-axis aberrations or changes ineye length as a function of retinal position can be post-processed afteracquisition to provide a sharper image over the entire field of view.Alternatively digital refocusing and/or aberration correction can beapplied after the individual volume images have been stitched togetherto form a single numerical representation of an optical characteristicof the retina over the combined volume. Variations in apparent eyelength are a common feature with myopic patients for example, whichwould normally limit the resolution of the image without adaptive opticsthat can track the acquisition. This information about the level ofdigital refocusing for off-axis aberration could also be clinicallymeaningful in assessment of myopia progression, as it provides aquantitative measure of some of the axial aberrations of the eye undertest. In visualising the layers of the retina or cornea, or of anon-ocular sample, it is often useful to do so as a B-scan wherein aslice of the sample is imaged. To enable this visualisation of highresolution detail in a B-scan, multiple adjoining volumes can beprocessed together encompassing the slice of interest, and the resultingthree-dimensional composite image reduced to a high resolution 13 scaneither through sampling or a weighted averaging of the area around theslice.

It will be appreciated that various elements in the FIG. 14 apparatusshould be operated in a coordinated fashion. These include for examplethe SLED 26, the sample arm polarisation control element 134, the beamsteering element 98, the angularly variable mirror 112 (or thetranslation stage 119) and the CMOS camera 6. This overall level ofcontrol may be provided, for example, by the processor 45 when equippedwith suitable machine-readable program code.

To enhance the lateral resolution and depth of field achievable with animaging system of given numerical aperture we now consider the casewhere for each set angle of the mirror 112 we take two or moremeasurements of the same volume 114 of the retina 89, illuminated ineach case with a different incident angle via different paths throughthe pupil 92 and an aperture 104 in the apertured reflector 102. In oneparticular example the apertured reflector has four apertures, with twoof the apertures separated at the extremes of the pupil in the verticalaxis and the other two apertures separated in the horizontal axis. Incertain embodiments the different illumination trajectories areestablished by angular adjustment of a 2-axis MEMS mirror 98 so thesample beam propagates sequentially through the specified apertures. Inother embodiments the different illumination trajectories areestablished simultaneously, e.g. with a diffractive optical element(DOE) as explained previously. In the former case the multiplemeasurements of the volume 114 are taken sequentially, i.e. single shotacquisition for each illumination trajectory (incident angle). In thelatter case the multiple measurements are taken simultaneously, i.e.single shot acquisition for all illumination trajectories. Either way,the far field captured for each of the different illuminationtrajectories therefore corresponds to angular offsets in the far field.In this manner, different regions of high frequency spatial content ofthe image, which would otherwise fall outside the NA of the system, havetheir frequency content shifted or ‘mixed’ down to baseband. Since eachillumination trajectory captures a different high frequency region, thecombined spatial content is potentially doubled compared with a singleillumination capture, thereby achieving a super resolution of half theRayleigh criterion. This approach is an improvement over Fourierptychography, described for example in Dong et al ‘Aperture-scanningFourier ptychography for 3D refocusing and super-resolution macroscopicimaging’, Optics Express 22(11), 13586-13599 (2014), in that the fieldis now captured interferometrically in a single snapshot rather thanhaving to be iteratively reconstructed to be consistent with theintensity image. With our approach the passband of the Fourier field canbe extended by registering and stitching together the different partialfar fields to create a stitched Fourier Plane, with each partial farfield acquired in a single shot. Fourier transformation or other digitalprocessing of the extended Fourier field measurements results in anenhanced lateral resolution. We do not have to rely on iterative methodsto infer what the field should have been based on intensity-onlymeasurements.

There are many situations where Doppler-like measurements of relativephase are of value, e.g. for measuring capillary blood flow or forperforming strain or elastography measurements in the presence of amechanical, acoustic thermo-acoustic or ultrasound perturbations. Tothis end, the apparatus shown in FIG. 4 or FIG. 10 for example can beadapted to provide a relative phase-sensitive OCT apparatus that can beutilised for Doppler-like measurements of motion or distortion, andrelated quantities such as strain, over a 3-D volume of a sample. Itwill be appreciated that other previously described apparatus can beadapted in similar fashion.

In this ‘Doppler’ embodiment the multi-wavelength optical source 26 istriggered to produce at least first and second optical pulses, each witha duration sufficiently short to allow a phase measurement to be made inthe presence of the motion or distortion which is to be measured withinthe interaction volume 19. The reflected or scattered light 18 from thefirst pulse is captured and analysed in a single exposure or frame ofthe 2-D sensor array 6 as has been described earlier. After apredetermined time period a second pulse is generated and its reflectedor scattered light 18 subsequently analysed in a second exposure of the2-D sensor array. Each exposure, after read-out and analysis, provides acomplex image comprising phase and amplitude information from theinteraction volume over a range of wavelengths. In certain embodimentsthe timing between the pulses is less than the frame rate of the sensorarray, which can be achieved by appropriately timing the pulsedillumination with respect to the exposures of the sensor array. That is,a first pulse can occur near the end of one frame, and a second pulsenear the beginning of the next frame. Obviously the pulsing of theoptical source 26 needs to be coordinated with operation of the sensorarray 6. In certain embodiments the optical source 26 is triggered bythe same processor 45 that reads out and analyses data from the sensorarray.

When using far field or Fourier plane sampling as shown in FIG. 4, thesample reflection spectra are analysed in the Fourier plane over anarray of locations corresponding to the sampling of the 2-D lensletarray 10. It is worth noting that for a given depth (corresponding to afrequency component of the spectral interferogram) within theinteraction volume 19 located approximately at the focal plane of thelens 22 there is, for each lateral sample location, a 2-D far fieldspatial frequency component. Furthermore a given specular reflection orspeckle frequency component will have a phase associated with it. Thelateral phase of a specific reflection is detected on the 2-D sensorarray 6 and can be retrieved during the numerical processing by theprocessor 45 by a complex FFT that returns both phase and amplitude ofreflection with spatial resolution, i.e. a complex image, over theinteraction volume 19. In regions where the amplitude is sufficientlystrong to provide a meaningful phase measurement, a set of points can beestablished over which the phase can be mapped. In certain embodimentsthe mapping constitutes just the axial component of the phase, obtainedby the FFT of the spectral components. In other embodiments the mappingalso comprises the transverse phase components, corresponding to thephase of the spatial far field components. The transverse phases need tobe scaled according to the focal length of the lens 22, and willtypically be a less sensitive measure of displacement. That is, a givenphase shift would represent a larger displacement than the axial phaseshift by a factor of around 3 to 10, depending on the numerical apertureof the captured light 18. Even allowing for the reduced sensitivity, forsubmicron displacements, this is still a valuable measurement that wouldbe difficult to achieve accurately using a conventional raster-scanningOCT system. It can therefore enable a more accurate measurement of thetrue velocity vector, i.e. rate of displacement, especially for caseswhere a capillary flow or other motion to be measured is largely in thetransverse plane and hence lacking an axial component.

If there is no bulk motion or distortion of the sample 20 between theillumination pulses, i.e. coarse movement much larger than theintra-sample motion or distortion of interest in the Dopplermeasurement, it is straightforward simply to subtract the relativephases of the data sets of points. However this is not always the case.It is particularly important for many measurements to be able toregister adequately the two frames of information corresponding to thetwo measurement sets. This bulk registration between the frames can beachieved by optimising a cross correlation function in the presence of agrid transformation that provides a given displacement and distortion(e.g. linear compression) mapping of the grid of the sample between oneframe and the next. Accurate registration of the two frames involvesaccounting for the phase shift associated with the mapping, to identifya basis from which to determine a relative phase shift caused by adisplacement associated with intra-sample motion (e.g. capillary flow)or distortion (e.g. mechanical perturbation) that is being determined.Knowledge of the predetermined time period between the frames enablesthe rate of displacement to be determined.

Elastography determines the local elasticity or stiffness of a sample,such as biological tissue, from displacement measurements. Localdisplacements, induced for example by compression of the sample, may beaccurately determined from relative phase measurements before and aftercompression. The local elasticity is inferred from the measureddisplacement as a function of depth. Alternatively, elasticity can bedetermined by using pulsed perturbations to generate low amplitude shearwaves, with the velocity and dispersion of these waves being sensitiveto the mechanical properties of the sample. Measuring the low amplitudesample displacements caused by the wave propagation requires theresolution offered by phase sensitive measurements.

It will be appreciated that the illustrated spectral domain OCT andlinear OCT embodiments, in which returning sample and reference beamsare sampled with a rectilinear 2-D lenslet array angled with respect tothe dispersive axis of a wavelength dispersive element, enable singleshot acquisition of 3-D images of a sample. In particular, theillustrated embodiments provide apparatus and methods for obtainingimproved high resolution optical images of a retina based on numericalreconstruction of the spectral characteristics of light reflected from asmall volume of the retina, with correction of aberrations present inthe sample eye.

In each of the illustrated embodiments, focusing of light beams isperformed with optical power elements in the form of lenses. However itwill be appreciated that other forms of optical power elements such asoff-axis parabolic or ellipsoidal mirrors could be used.

Although the invention has been described with reference to specificexamples, it will be appreciated by those skilled in the art that theinvention may be embodied in many other forms.

The claims defining the invention are as follows:
 1. A high resolutionoptical imaging apparatus, comprising: (i) an illumination system forilluminating, with a multi-wavelength optical beam, a volume of a sampleto be imaged in three spatial dimensions; (ii) a sampling system forsampling in the image plane light reflected or scattered from theilluminated volume of said sample; (iii) a measurement system forsimultaneous capture of phase and amplitude information over a range ofwavelengths of the sampled reflected or scattered light; and (iv) aprocessor for processing the phase and amplitude information toconstruct a three-dimensional image of an optical characteristic of saidsample over said illuminated volume, wherein said optical characteristicis selected from the group consisting of: phase, reflectivity,refractive index, refractive index changes and attenuation.
 2. Anapparatus according to claim 1, wherein said processor is adapted toconstruct said three-dimensional image using digital refocusing ordigital correction of aberrations of said sample.
 3. An apparatusaccording to claim 1, wherein said measurement system comprises awavelength dispersive element for dispersing the sampled signalsobtained from said sampling system onto a two-dimensional sensor array,wherein said sampling system is positioned with respect to saidwavelength dispersive element such that, in use, each of said sampledsignals is dispersed onto a set of pixels of said sensor array.
 4. Anapparatus according to claim 1, wherein said sampling system comprises atwo-dimensional lenslet array for sampling the reflected or scatteredlight to provide a two-dimensional grid of sampling points.
 5. Anapparatus according to claim 1, wherein the illuminated surfacecorresponding to said illuminated volume is less than or equal to 500μm×500 μm in area, more preferably less than or equal to 200 μm×200 μmin area.
 6. An apparatus according to claim 1, wherein saidthree-dimensional image has a spatial resolution of 3 μm or better. 7.An article of manufacture comprising a computer usable medium having acomputer readable program code configured to operate the apparatusaccording to claim
 1. 8. A method for performing high resolution opticalimaging of a sample, said method comprising the steps of: (i)illuminating, with a multi-wavelength optical beam, a volume of a sampleto be imaged in three spatial dimensions; (ii) sampling in the imageplane light reflected or scattered from the illuminated volume of saidsample; (iii) simultaneously capturing phase and amplitude informationover a range of wavelengths of the sampled reflected or scattered light;and (iv) processing the phase and amplitude information to construct athree-dimensional image of an optical characteristic of said sample oversaid illuminated volume, wherein said optical characteristic is selectedfrom the group consisting of: phase, reflectivity, refractive index,refractive index changes and attenuation.
 9. A method according to claim8, wherein said three-dimensional image is constructed using digitalrefocusing or digital correction of aberrations of said sample.
 10. Amethod according to claim 8, wherein the signals obtained from thesampling in the image plane are dispersed with a wavelength dispersiveelement onto a two-dimensional sensor array, wherein the sampling systemis positioned with respect to said wavelength dispersive element suchthat each of said signals is dispersed onto a set of pixels of a sensorarray.
 11. A method according to claim 8, wherein the light reflected orscattered from the illuminated volume of said sample is sampled in theimage plane using a two-dimensional lenslet array.
 12. A methodaccording to claim 8, wherein the illuminated surface corresponding tosaid volume is less than or equal to 500 μm×500 μm in area, morepreferably less than or equal to 200 μm×200 μm in area.
 13. A methodaccording to claim 8, wherein said three-dimensional image has a spatialresolution of 3 μm or better.
 14. An article of manufacture comprising acomputer usable medium having a computer readable program codeconfigured to implement the method according to claim 8.